Hemostasis is maintained by a complex interplay of a variety of enzymes. Clotting factors interact in a "cascade" of activation steps which eventually leads to the formation of a fibrin clot. Subsequent degradation of the fibrin clot is accomplished by the fibrinolytic system, which involves the serine protease plasmin, a proteolytic enzyme which breaks down fibrin plasmin is a broad spectrum protease which also cleaves certain coagulation factors, thereby inactivating them. Production of plasmin from its inactive precursor, plasminogen, is mediated by tissue plasminogen activator (t-PA), itself a serine protease. The proteolytic activity of plasmin on fibrin clots is inhibited by .alpha..sub.2 -plasmin inhibitor (.alpha..sub.2 -PI). .alpha..sub.2 -PI is covalently attached to the fibrin matrix through the action of factor XIIIa, a clot stabilizing enzyme (Sakata and Aoki, J. Clin Invest. 69:536-542, 1981; Tamaki and Aoki, Biochem. Biophys Acta 661:280-286, 1981; Aoki et al., J. Clin Invest. 63:877-884, 1979; and Tamaki and Aoki, J. Biol. Chem. 257:14767-14772, 1982).
If the normal hemostatic system becomes upset, clots may form at inappropriate times and places, leading to myocardial infarction, deep vein thrombosis, pulmonary embolism and stroke. Tissue damage resulting from these conditions may result in death or serious disability.
Tissue damage does not occur immediately upon obstruction of the blood supply to tissues, but develops over several hours after the initial stoppage of blood flow. Accordingly, the use of fibrinolytic agents to restore blood flow and minimize damage is of great clinical interest.
Three fibrinolytic agents are now in use. These are streptokinase, a bacterial protein; urokinase, a serine protease originally found in human urine; and tissue plasminogen activator, the natural activator of plasminogen. Streptokinase is an activator of plasminogen, able to convert the zymogen to its active form throughout the body. This may lead to non-specific plasminogen activation and to disruption of the hemostatic system as coagulation factors are degraded. Furthermore, streptokinase is a foreign protein and may therefore elicit an immune response in patients receiving it. Urokinase, although a human protein, is also not specific for activity at the site of the clot. Tissue plasminogen activator has been viewed as the preferred agent for fibrinolytic therapy because it is the normal vascular activator of plasminogen and because its activity is stimulated in the presence of fibrin. Early studies indicated that therapeutic administration of t-PA could result in lysis of undesirable clots without the systemic effects seen with streptokinase and urokinase. However, as will be discussed below, native t-PA has not fulfilled these expectations in clinical trials.
t-PA normally circulates in the blood as a single polypeptide chain of Mr=72,000 daltons which is converted to a two chain form by cleavage of a peptide bond between amino acids 275 (Arg) and 276 (Ile) (numbering of t-PA amino acids is shown in FIG. 1). The heavy chain of t-PA (two variants of Mr 40,000 and 37,000) is derived from the amino terminus of the protein and is responsible for the binding of t-PA to fibrin, while the light chain (Mr 33,000) is derived from the carboxy-terminal end of the molecule and contains the serine protease domain. The structure of t-PA is reviewed by Ny et al. (Proc. Natl. Acad. Sci. USA 81:5355-5359, 1984.) Cleavage to the two chain form is catalyzed by trypsin or plasmin, and is accompanied by an increase in activity as measured using synthetic substrates, and by an increase in fibrin-independent proteolytic activity. Binding of t-PA to fibrin facilitates this cleavage. t-PA binds to fibrin in a non-covalent manner at two sites in the heavy chain of the activator. The finger domain and the kringle 2 domain bind to fibrin with high affinity (van Zonneveld et al., J. Biol. Chem. 261:14212, 1986). Binding at the kringle 2 domain occurs via a lysine binding site on that structure.
The single chain form of t-PA is essentially inactive in the absence of fibrin. However, once it is converted to the two chain form, it also possesses considerable proteolytic activity in the absence of polymerized fibrin and may circulate through the blood stream and activate plasminogen in a nonspecific manner. This effect becomes significant in a clinical situation where doses of t-PA far greater than normal physiological levels must be administered. The short plasma half-life of t-PA and the presence of circulating t-PA inhibitors add to the need for large doses. In practice, then, it has been found that t-PA shares many of the undesirable side effects of streptokinase and urokinase.
Various researchers have attempted to alter t-PA to increase its fibrin affinity or specificity. Rosa and Rosa (published PCT application WO 86/01538) modified the Lys at position 277 of native t-PA to stabilize the single chain form of the molecule. Heyneker and Vehar (published UK patent application 2,173,804) produced variant forms of t-PA having amino acid substitutions around the cleavage site. Van Zonneveld et al. (Proc. Natl. Acad. Sci. USA 83:4670-4674, 1986) and Robinson et al. (EP 207,589) disclose mutant forms of t-PA in which portions of the heavy chain have been deleted. Hung et al. (published UK patent application 2,179,948) disclose plasminogen activators containing plural, heterologous kringle domains. Haigwood et al. (EP 227,462) mutagenized t-PA at several sites in an effort to improve its properties. These variant forms of t-PA do not fully overcome the problems associated with the native molecule, nor has their efficacy been demonstrated in a clinical setting.
There remains a need in the art for a plasminogen activating protein with an enhanced affinity for fibrin and a consequent increase in clot lysing specificity. The present invention fulfills this need by providing novel hybrid proteins derived from tissue plasminogen activator and a protein which can be cross-linked to fibrin by factor XIIIa. A particularly preferred such protein is .alpha..sub.2 -plasmin inhibitor. These hybrid proteins have an increased affinity for fibrin due to their ability to be covalently cross-linked to fibrin by factor XIIIa. The resulting increase in specificity leads to a reduction of systemic bleeding effects and greater clinical suitability. The hybrid proteins can be used to lyse existing clots in heart attack and stroke victims and in others where specific clot lysis is therapeutically desirable. In addition, they may be used as diagnostic agents for imaging blood clots.